Critical role of conserved proline residues in the transmembrane segment 4voltage sensor function and in the gating of L-type calcium channels

The fourth transmembrane segment (S4) has been shown to function as a volta ge sensor in voltage-gated channels. On membrane depolarization, a stretch of S4 moves outward and initiates a number of conformational changes that u ltimately lead to channel opening. Conserved proline residues are in the mi ddle of the S4 of motifs I and III in voltage-dependent Ca2+ channels. Beca use proline often introduces a "kink" into a helical structure of proteins, these residues might have an intrinsic function in the voltage sensor. Her e, we report that the removal of S4 prolines results in a dramatic shorteni ng of channel open time whereas the introduction of extra prolines to the c orresponding positions in motif IIS4 and IVS4 lengthens channel open time. The number of S4s with a proline residue showed a clear positive correlatio n with the mean open time of the channel. The mean open time was >11-fold l onger for a channel mutagenized to have prolines in all four S4s compared w ith a channel that had no prolines in the S4 region. Additionally, prolines in the S4s slowed activation kinetics and shifted the voltage dependence o f activation and inactivation in a hyperpolarized direction. Our results st rongly suggest that proline residues in the S4s are critical for stabilizin g the open state of the channel. Moreover, it is suggested that motif IS4 a nd IIIS4 contribute to the channel opening more efficiently than motif IIS4 and IVS4.